Genetic modifications in bacteria for the degradation of synthetic polymers: a review

Citation: Martín-González, D.; de la Fuente Tagarro, C.; De Lucas, A.; Bordel, S.; Santos-Beneit, F. Genetic Modifications in Bacteria for the Degradation of Synthetic Polymers: A Review. Int. J. Mol. Sci. 2024,25, 5536. https://doi.org/10.3390/ ijms25105536 Academic Editor: Jordi Puiggalí Received: 28 March 2024 Revised: 7 May 2024 Accepted: 17 May 2024 Published: 19 May 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Review Genetic Modifications in Bacteria for the Degradation of Synthetic Polymers: A Review Diego Martín-González 1,† , Carlos de la Fuente Tagarro 1,†, Andrea De Lucas 1, Sergio Bordel 1,2 and Fernando Santos-Beneit 1,2,* 1Department of Chemical Engineering and Environmental Technology, School of Industrial Engineering, University of Valladolid, Dr. Mergelina, s/n, 47011 Valladolid, Spain; [email protected] (D.M.-G.); [email protected] (A.D.L.); sergio.bor[email protected] (S.B.) 2Institute of Sustainable Processes, Dr. Mergelina s/n, 47011 Valladolid, Spain *Correspondence: [email protected] †These authors contributed equally to this work. Abstract: Synthetic polymers, commonly known as plastics, are currently present in all aspects of our lives. Although they are useful, they present the problem of what to do with them after their lifespan. There are currently mechanical and chemical methods to treat plastics, but these are methods that, among other disadvantages, can be expensive in terms of energy or produce polluting gases. A more environmentally friendly alternative is recycling, although this practice is not widespread. Based on the practice of the so-called circular economy, many studies are focused on the biodegradation of these polymers by enzymes. Using enzymes is a harmless method that can also generate substances with high added value. Novel and enhanced plastic-degrading enzymes have been obtained by modifying the amino acid sequence of existing ones, especially on their active site, using a wide variety of genetic approaches. Currently, many studies focus on the common aim of achieving strains with greater hydrolytic activity toward a different range of plastic polymers. Although in most cases the depolymerization rate is improved, more research is required to develop effective biodegradation strategies for plastic recycling or upcycling. This review focuses on a compilation and discussion of the most important research outcomes carried out on microbial biotechnology to degrade and recycle plastics. Keywords: synthetic polymers; plastics; biodegradation; genetic engineering; PET; PETase; cutinase; esterase 1. Introduction 1.1. Definition and Classification of Plastics Plastics are a type of synthetic polymeric material based on carbon and hydrogen and with high molecular weight [ 1 ]. Industrial-scale plastic production began in the 20th Century, and their use has increased over time [ 2 , 3 ]. Plastics can be classified according to different criteria. For example, depending on the composition of their backbone, they can be homochain polymers (when their backbone is made up exclusively of carbon) or heterochain polymers (if there are other elements, like oxygen and nitrogen, present in their backbone) [ 4 ]. Depending on the monomers that they are made of, they can be classified as homopolymers if they only have one monomer or copolymers if they have two or more different monomers [ 1 ]. Depending on their thermomechanical properties, they can be thermosets if, during their fabrication, covalent bonds are established between polymer chains, giving them high resistance and a shape that cannot be modified thermically; thermoplastics, if no covalent bonds are established between polymer chains so they can be fused and reshaped; or elastomers if they have a high elasticity [ 5 ]. Depending on the presence of benzene rings in their backbone, they can be aromatic if they have benzene rings or aliphatic if they do not have benzene rings [ 6 ]. Depending on the raw material Int. J. Mol. Sci. 2024,25, 5536. https://doi.org/10.3390/ijms25105536 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2024,25, 5536 2 of 22 they are made from, they can be petrochemical (the raw material is petroleum) or biobased (the raw material is biomass), and depending on their degradability by organisms, they can be biodegradable or non-biodegradable. 1.2. Advantages and Disadvantages of Plastics In general, plastics are lightweight, long-lasting, inert and cheap and easy to produce. Their diversity is such that there are plastics with the ideal characteristics for almost any application. Furthermore, the use of mixtures and alloys, as well as additives, can change the properties of the material and adjust them as desired [ 1 ]. As plastics are remarkably diverse and can have vastly different properties, their uses are just as varied. They are used to make, among many others, fibers and textiles, toys, packaging, healthcare instruments, such as syringes and implants, and construction materials for insulation, pipes and cable coatings [ 1 ]. Even though plastics are generally cheap and easy to produce and have good mechanical properties, the field of engineering requires especially resistant materials for very specific applications. Though they are more expensive, the so-called “engineering plastics” are used for that purpose as they boast a high performance [ 1 ]. Other plastics, known as commodity plastics, are found in everyday items. The most used commodity plastics are poly(ethylene terephthalate) (PET), highdensity polyethylene (HDPE), poly(vinylchloride) (PVC), low-density polyethylene (LDPE), polypropylene (PP) and polystyrene (PS) [ 2 , 7 , 8 ]. Products made with these plastics can be identified according to the ASTM International Resin Identification Coding System (RIC). The “Others” category includes all other plastics, as well as products made with a mixture of two or more plastics [9]. The global annual production of plastics exceeded 400 million metric tonnes (400 Mt) in 2022, 362.3 of which were new petrochemical plastics. Commodity plastics make up the following percentages (see Figure 1): PET 6.2%, HDPE 12.2%, PVC 12.7%, LDPE 14.1%, PP 18.9% and PS 5.2% [ 10 ]. Out of the different uses, most plastic is destined for packaging. In 2019, 142 Mt (31%) of plastics were used in packaging [ 3 , 7 ]. Between 1950 and 2022, more than 10 billion metric tonnes (10.000 Mt) of plastics were produced [2,10,11]. Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 2 of 23 they have benzene rings or aliphatic if they do not have benzene rings [6]. Depending on the raw material they are made from, they can be petrochemical (the raw material is petroleum) or biobased (the raw material is biomass), and depending on their degradability by organisms, they can be biodegradable or non-biodegradable. 1.2. Advantages and Disadvantages of Plastics In general, plastics are lightweight, long-lasting, inert and cheap and easy to produce. Their diversity is such that there are plastics with the ideal characteristics for almost any application. Furthermore, the use of mixtures and alloys, as well as additives, can change the properties of the material and adjust them as desired [1]. As plastics are remarkably diverse and can have vastly different properties, their uses are just as varied. They are used to make, among many others, fibers and textiles, toys, packaging, healthcare instruments, such as syringes and implants, and construction materials for insulation, pipes and cable coatings [1]. Even though plastics are generally cheap and easy to produce and have good mechanical properties, the field of engineering requires especially resistant materials for very specific applications. Though they are more expensive, the so-called “engineering plastics” are used for that purpose as they boast a high performance [1]. Other plastics, known as commodity plastics, are found in everyday items. The most used commodity plastics are poly(ethylene terephthalate) (PET), highdensity polyethylene (HDPE), poly(vinylchloride) (PVC), low-density polyethylene (LDPE), polypropylene (PP) and polystyrene (PS) [2,7,8]. Products made with these plastics can be identified according to the ASTM International Resin Identification Coding System (RIC). The “Others” category includes all other plastics, as well as products made with a mixture of two or more plastics [9]. The global annual production of plastics exceeded 400 million metric tonnes (400 Mt) in 2022, 362.3 of which were new petrochemical plastics. Commodity plastics make up the following percentages (see Figure 1): PET 6.2%, HDPE 12.2%, PVC 12.7%, LDPE 14.1%, PP 18.9% and PS 5.2% [10]. Out of the different uses, most plastic is destined for packaging. In 2019, 142 Mt (31%) of plastics were used in packaging [3,7]. Between 1950 and 2022, more than 10 billion metric tonnes (10.000 Mt) of plastics were produced [2,10,11]. Figure 1. Global production of the 6 commodity plastics (in million metric tons). Data obtained from [10]. Despite their useful properties, the use of plastics has two main disadvantages, which have to do with the beginning and the end of their lifetime: the first one is the fact that most plastics are petrochemical, and the second one is the amount of non-biodegradable waste generated. Regarding their origin, the reserves of petroleum are limited as petroleum is a non-renewable resource, and given the production rate in 2020, it is Figure 1. Global production of the 6 commodity plastics (in million metric tons). Data obtained from [10]. Despite their useful properties, the use of plastics has two main disadvantages, which have to do with the beginning and the end of their lifetime: the first one is the fact that most plastics are petrochemical, and the second one is the amount of non-biodegradable waste generated. Regarding their origin, the reserves of petroleum are limited as petroleum is a non-renewable resource, and given the production rate in 2020, it is estimated that reserves will last for 50 years [ 12 ]. For the purposes of this article, the waste generated is the most relevant problem. Int. J. Mol. Sci. 2024,25, 5536 3 of 22 2. Bioplastics as an Alternative to Petroleum-Based and Non-Biodegradable Plastics The most recent assessments estimate that out of the 9200 Mt of plastics produced until 2017, 6500 Mt (70%) have become waste. Of this waste, 5000 Mt (54%) ended up in landfills or were released into the environment [ 13 ]. This plastic waste has reached virtually every corner of the planet. The five so-called garbage patches are notorious for accumulating 250,000 tons of plastic [ 7 ], but plastics are also found elsewhere. They have been found in rivers, lakes, drinking water and table salt and even in extremely remote places, such as the seabed and both polar regions. They have reached such areas mostly in the form of microand nanoplastics, whose size allows them to travel much further and even enter organisms’ cells [ 14 ]. Due to their composition and structure, plastics can remain in the environment for thousands of years [ 15 ], and their effects on living beings’ health, while a complicated matter that is still under study, are undoubtedly severely negative. This has been researched especially in marine ecosystems, where they can cause harm to many different organisms [ 8 , 16 , 17 ], but they are also apparent in terrestrial ecosystems [ 18 ] and human health [ 19 , 20 ]. Moreover, the additives used to modify the properties of plastics can also have toxic effects on organisms [ 21 ], and plastics can adsorb and accumulate heavy metals, persistent organic contaminants and even pathogens [22]. Due to their harmful effects on the environment, plastics, like any other type of waste, have to be managed appropriately. Recycling rates are overall very low, with only 600 Mt (6.5%) of plastics produced worldwide until 2017 having been recycled and more than 5000 Mt of them ending up in landfills or being released into the environment [ 13 ]. Landfilling plastic waste, though simple, is not an adequate waste management strategy, as landfills can leach microplastics [ 23 , 24 ] as well as other contaminants [ 25 ]. Moreover, plastic waste in the environment does not remain permanently unchanged, as it slowly undergoes physicochemical changes that partially break it down. These changes include photodegradation, hydrolysis and thermo-oxidation. However, this transformation results mostly in the fragmentation of plastic rather than its mineralization, generating smaller plastic fragments (microand nanoplastics) that still remain in the environment. In order for plastics to be mineralized and, therefore, completely removed from the environment, biological degradation is required [ 26 ]. This biological degradation can be performed by different organisms but, even if plastics end up mineralized by the action of these organisms, the degradation rates are so slow and the plastic permanence is so high that most plastics are considered non-biodegradable. Given the problems derived from the properties of the most used plastics, a new type of material has received a lot of attention: bioplastics. Bioplastics are a type of plastic that differs from conventional plastic in at least one of two ways: the raw material they are made from or their biodegradability. Bioplastics can either be made from biomass—biobased plastics—or be biodegradable or have both properties [ 27 ]. A particular type of bioplastics is poly(hydroxyalkanoates) (PHAs). They are naturally produced by some microorganisms, mostly bacteria, for energy storage [ 28 ] and are both biobased (as they are produced by an organism that can be grown with renewable feedstock) [ 29 ] and biodegradable (as they can be easily enzymatically degraded) [ 30 ]. Just like plastics in general, bioplastics are very varied, so grouping them in the bioplastic category is not particularly informative of their properties, especially without specifying which criteria make them such. Therefore, when discussing individual plastics, this work will specify if they are petrochemical or biobased and biodegradable or non-biodegradable (Figure 2). The main benefits of using bioplastics are summarized in the fact that biobased plastics do not involve the use of petroleum, and biodegradable plastics can be fully mineralized by organisms. However, bioplastic production still has an environmental impact when the whole life cycle is considered [ 31 ], and regarding biodegradable plastics, this property does not mean that they can be released into the environment without consequences [32]. Int. J. Mol. Sci. 2024,25, 5536 4 of 22 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 of 23 biodegradable plastics, this property does not mean that they can be released into the environment without consequences [32]. Figure 2. Classification of plastics according to the raw material and biodegradability. PHAs (including a major type, i.e. PHBV) constitute a special category of plastics since they are completely natural. Biodegradable plastics can remain in the environment for long periods of time [33– 36] and carry potentially toxic substances [37]. They can even act as a reservoir of microbes with antibiotic-resistance genes [38], so their waste should be managed, too [39]. In the case of biodegradable plastics, the preferred waste management strategy is carrying out their degradation by organisms. There are international standards developed to clearly define the conditions and timescale of biodegradation [40–46], but their use is not mandatory, so the criteria to determine the biodegradability of plastics can be quite arbitrary. However, plastic is generally considered biodegradable if there are organisms that can mineralize it in a reasonable amount of time in controlled conditions, which must always be specified. 3. Biodegradation of Plastic Polymers The last few decades have seen an increasing interest in both biodegradable plastics and organisms that can degrade plastics. As new species and strains are discovered and genetic engineering is used to improve the enzymes involved in biodegradation, plastics that were previously considered non-biodegradable can now be degraded by one organism or another and to a greater or lesser extent. Therefore, the categories of biodegradable and non-biodegradable plastics are not fixed and can change over time, but in this work, we will use the usual classification. Plastic-degrading microorganisms include bacteria, fungi and microalgae [47]. This review focuses on bacterial strains. Although genetic engineering approaches have been performed for decades, only a few bacteria have been modified to improve their capability of degrading these synthetic plastics or to grant them this characteristic by modifying their metabolism. More specifically, the modifications performed consist of the heterologous expression of enzymes that come from other organisms, mutating/changing specific amino acids of the enzymes and the addition of domains or other protein structures to the enzymes. Different techniques have been used in order to carry out these modifications, Figure 2. Classification of plastics according to the raw material and biodegradability. PHAs (including a major type, i.e., PHBV) constitute a special category of plastics since they are completely natural. Biodegradable plastics can remain in the environment for long periods of time [ 33 – 36 ] and carry potentially toxic substances [ 37 ]. They can even act as a reservoir of microbes with antibiotic-resistance genes [ 38 ], so their waste should be managed, too [ 39 ]. In the case of biodegradable plastics, the preferred waste management strategy is carrying out their degradation by organisms. There are international standards developed to clearly define the conditions and timescale of biodegradation [ 40 – 46 ], but their use is not mandatory, so the criteria to determine the biodegradability of plastics can be quite arbitrary. However, plastic is generally considered biodegradable if there are organisms that can mineralize it in a reasonable amount of time in controlled conditions, which must always be specified. 3. Biodegradation of Plastic Polymers The last few decades have seen an increasing interest in both biodegradable plastics and organisms that can degrade plastics. As new species and strains are discovered and genetic engineering is used to improve the enzymes involved in biodegradation, plastics that were previously considered non-biodegradable can now be degraded by one organism or another and to a greater or lesser extent. Therefore, the categories of biodegradable and non-biodegradable plastics are not fixed and can change over time, but in this work, we will use the usual classification. Plastic-degrading microorganisms include bacteria, fungi and microalgae [ 47 ]. This review focuses on bacterial strains. Although genetic engineering approaches have been performed for decades, only a few bacteria have been modified to improve their capability of degrading these synthetic plastics or to grant them this characteristic by modifying their metabolism. More specifically, the modifications performed consist of the heterologous expression of enzymes that come from other organisms, mutating/changing specific amino acids of the enzymes and the addition of domains or other protein structures to the enzymes. Different techniques have been used in order to carry out these modifications, such as metagenomic analysis, directed and non-directed mutagenesis and the use of fusion proteins or chimeras. This review is a compilation of the studies in which bacteria have been genetically modified, whether to grant them or enhance their ability to degrade plastics or simply to perform genetic modifications and will only mention the polymers for which modified Int. J. Mol. Sci. 2024,25, 5536 5 of 22 bacteria have been obtained. Table 1shows the modified bacterial strains that have been developed for the degradation of plastic polymers according to research published in the scientific literature (i.e., PubMed, Scopus, etc.). Table 1. Heterologous expression of enzymes to degrade PET, PLA, PBAT, PVAC, PCL, PBS, PHV, PU and LMWPE. Host Plastic Enzyme Origin Species Ref. E. coli BL21 (DE3) PLA Protease (Plasmid: pET26b(+)) Thermus sp. Rt41A [48] E. coli BL21-Gold (DE3) PBAT Esterases Cbotu_EstA and Cbotu_EstB Clostridium botulinum [49] E. coli DH5αPVAC, PCL Cutinase (Cut) and lipase (Lip) (Plasmid: pPICZαA) Thermomyces lanuginosus (Lip); Thielavia terrestris NRRL 8126 (Cut) [50] E. coli XL-10 PET, PBS, PHBV Cutinase 1 (Thc_Cut1) (Plasmid: pMK-T; pPICZαB) Thermofida cellulosilytica [51] E. coli XL10-Gold PU Polyamidase (PA) (Plasmid: pET26b(+)) Nocardia farcinica IMA 10152A (PA) [52] E. coli BL21 Gold (DE3) PU Polyamidase (PA) (Plasmid: pET26b(+)) Nocardia farcinica IMA 10152A (PA) [52] E. coli BL21 LMWPE Alkane hydroxylase (Plasmid: pUC19) Pseudomonas sp. E4 [53] E. coli BL21-CodonPlus (DE3) PET, PCL Cutinase Metagenomical library [54] Since PET is one of the most produced plastics worldwide and its degradation is challenging, a special focus is put in this review in relation to the microbial biodegradation strategies developed specifically for this polymer (summarized in Table 2). Table 2. Heterologous expression of enzymes to degrade PET. Host Plastic Enzyme Origin Species Ref. E. coli BL21 (DE3) PET Cutinase (Plasmid: pET25b(+)) Fusarium solani pisi [55] E. coli BL21 (DE3) PET Cutinase Tfu_0883 (Plasmid: pET20b) Thermobifida fusca [56] E. coli BL21-Gold (DE3) PET Cutinase Thc_Cut2 (Plasmid: pET26b(+)) Thermobifida cellulosilytica DSM44535 [57] E. coli DH5αPET Cutinase-type polyesterase (Cut190) (Plasmid: pGEM-T; pQE80L) Saccharomonospora viridis AHK190 [58] E. coli Rosetta-gami B (DE3) PET Cutinase-type polyesterase (Cut190) (Plasmid: pGEM-T; pQE80L) Saccharomonospora viridis AHK190 [58] E. coli BL21 (DE3) PET Cutinase TfCut2 Thermobifida fusca KW3 [59] E. coli BL21 (DE3) PET Cutinase TfCut2, LC-Cutinase, carboxyl esterase TfCa (Plasmid: pET-20b(+)) Thermobifida fusca KW3 [60] E. coli XL1-Blue PET PETase (Plasmid: pET32a) Ideonella sakaiensis 201-F6 [61] E. coli BL21 DE3 PET LC-cutinase (Plasmid: PET28; PJ912) Plant compost metagenome [62] Int. J. Mol. Sci. 2024,25, 5536 6 of 22 Table 2. Cont. Host Plastic Enzyme Origin Species Ref. E. coli Rosetta-gami B PET PETase (Plasmid: pET15b; pET15a) Ideonella sakaiensis [63] E. coli BL21-CodonPlus (DE3) RIPL PET PETase (Plasmid: pET-21b) Ideonella sakaiensis [64] E. coli C41 (DE3) PET PETase (Plasmid: pET-21b(+)) Ideonella sakaiensis 201-F6 [65] E. coli BL21 (DE3) PET PETase (Plasmid: pET28a) Ideonella sakaiensis [66] E. coli Rosetta-gami B PET Multiple modified IsPETase (IsPETaseS121E/D186H/R280A) Ideonella sakaiensis [67] E. coli DH5αPET LC cutinase (Plasmid: pHK-LCC) Plant compost metagenome [68] E. coli BL21 (DE3) PET LC cutinase (Plasmid: pHK-LCC) Plant compost metagenome [68] Clostridium thermocellum DSM1313 PET LC cutinase (Plasmid: pHK-LCC) Plant compost metagenome [68] E. coli BL21 (DE3) PET Hydrolases 1 and 2 (BTA1 and BTA2); cutinase (FsC); IsPETase; leaf-branch compost cutinase (LCC) Thermobifida fusca (BTA1 and BTA2); Fusarium solani pisi (FsC); Ideonella sakaiensis 201-F6 (IsPETase); leaf compost metagenome (LCC) [69] E. coli MG1655 RARE PET Terephthalate 1,2-dioxygenase, dihydroxy-3,5-cyclohexadiene1,4-dicarboxylic acid dehydrogenase, carboxylic acid reductase, catechol O-methyltransferase Ideonella sakaiensis [70] E. coli PHL628 PET PETase bound to BIND platform Ideonella sakaiensis [71] E. coli TOP10 PET PETase bound to BIND platform Ideonella sakaiensis [71] E. coli BL21 (DE3) PET Leaf-branch compost, cutinase (LCC) and variants Leaf compost metagenome [72] E. coli DH5αPET Leaf-branch compost, cutinase (LCC) and variants Leaf compost metagenome [72] E. coli NEB5αPET IsPETase; IsPETase-MHETase chimera Ideonella sakaiensis [73] Vibrio natriegens PET IsPETase; IsPETase-MHETase chimera Ideonella sakaiensis [73] 3.1. Biodegradation of PET PET is one of the most produced petrochemical synthetic polymers, accounting for around 6.2% of global plastic production in 2022 [ 10 ], and its market size surpasses other highly produced plastics, such as HDPE, PVC or PP [ 74 ]. The main reason why PET is so produced is because its molecular structure offers a lot of versatility, making it essential in our daily lives. PET is a long semi-aromatic thermoplastic polyester chain produced from ethylene glycol (EG) and terephthalic acid (TPA). Its production has two steps. First, the union of two EG molecules and one TPA molecule by esterification generates an intermediate molecule, bis(2-hydroxyethyl) terephthalate (BHET). Secondly, BHET along with catalysts, such as Sb 2 O 3 or Sb(OAc) 3 , is subjected to a process of polymerization, Int. J. Mol. Sci. 2024,25, 5536 7 of 22 creating the long chain through ester bonds (Figure 3A) [ 5 , 75 ]. When it comes to the manufacturing process, amorphous and semi-crystalline PET are produced depending on the thermal processing undergone during the polymerization. The main difference between these two types lies in the intrinsic viscosity and molecular weight. In the case of the amorphous polymer, the long polyester chains are randomly set out, resulting in a more flexible plastic. On the other hand, semi-crystalline materials are formed by amorphous domains and chains arranged in an orderly way, making the material more resistant and less ductile (Figure 3B). These differences in its molecular structure make it both chemically and thermally stable. This is what makes PET a strong and durable compound, ideal for a wide variety of applications, such as synthetic fibers for the textile industry, water bottles and packaging [5,76]. Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 23 between these two types lies in the intrinsic viscosity and molecular weight. In the case of the amorphous polymer, the long polyester chains are randomly set out, resulting in a more flexible plastic. On the other hand, semi-crystalline materials are formed by amorphous domains and chains arranged in an orderly way, making the material more resistant and less ductile (Figure 3B). These differences in its molecular structure make it both chemically and thermally stable. This is what makes PET a strong and durable compound, ideal for a wide variety of applications, such as synthetic fibers for the textile industry, water bottles and packaging [5,76]. Figure 3. (A) PET industrial formation process using BHET as intermediate. Based on [5]. (B) Molecular structure of amorphous (up) and semi-crystalline (down) materials of PET. Adapted from [77]. Despite the advantages provided by PET, it also presents the serious drawback of what to do with it after its useful life. Most PET comes to an end accumulated in landfills. It is estimated that it takes around 300 years to decompose, degrading over time because of solar radiation and heat, among other factors, and releasing harmful chemical compounds to the environment. The estimate increases if its degradation is not accelerated by heat or solar radiation, reaching 2500 years or more. PET is so difficult to degrade due to its physicochemical properties, which make it resistant to decomposition by water and organic and inorganic compounds [73,76]. For this reason, there are a series of methods, which involve mechanical, chemical and biological methods, to recycle and reuse this polymer, producing fibers and fabrics. The mechanical methods are the most widespread, but they are also very expensive, while in the case of chemical methods, the process can be harmful to the environment [70,78]. In contrast with the previously mentioned methods, biological methods are on the rise because they do not damage the environment. These methods are still in development and are based on the use of cells as factories of enzymes able to break the bonds of PET, releasing the monomers of which it is composed. After that, those different monomers can be subjected to a valorization process to generate high value-added molecules, such as polyhydroxyalkanoates, vanillic acid, gallic acid, lycopene, glycolic acid, pyrogallol, catechol and muconic acid, which can be used as flavors, cosmetics, sanitizers and animal feed and in pharmacy, among other uses [78,79]. 3.1.1. Enzymes Involved in the PET Degradation Pathway The reason why PET can be degraded by enzymes is because a lot of them are unspecific regarding their substrates. The current literature offers many names given to these enzymes, such as PET hydrolases, PET esterases, PET cutinases, PET depolymerases or PETases. PET esterases, PET cutinases and PET depolymerases are hydrolytic enzymes that are able to break the ester bonds of biological molecules, like suberin and cutin, and due to their unspecificity, it turns out that they can break those of PET as well. On the other hand, PETases are hydrolytic enzymes more specific to PET [80,81]. Due to the Figure 3. (A) PET industrial formation process using BHET as intermediate. Based on [ 5 ]. (B) Molecular structure of amorphous (up) and semi-crystalline (down) materials of PET. Adapted from [77]. Despite the advantages provided by PET, it also presents the serious drawback of what to do with it after its useful life. Most PET comes to an end accumulated in landfills. It is estimated that it takes around 300 years to decompose, degrading over time because of solar radiation and heat, among other factors, and releasing harmful chemical compounds to the environment. The estimate increases if its degradation is not accelerated by heat or solar radiation, reaching 2500 years or more. PET is so difficult to degrade due to its physicochemical properties, which make it resistant to decomposition by water and organic and inorganic compounds [73,76]. For this reason, there are a series of methods, which involve mechanical, chemical and biological methods, to recycle and reuse this polymer, producing fibers and fabrics. The mechanical methods are the most widespread, but they are also very expensive, while in the case of chemical methods, the process can be harmful to the environment [70,78]. In contrast with the previously mentioned methods, biological methods are on the rise because they do not damage the environment. These methods are still in development and are based on the use of cells as factories of enzymes able to break the bonds of PET, releasing the monomers of which it is composed. After that, those different monomers can be subjected to a valorization process to generate high value-added molecules, such as polyhydroxyalkanoates, vanillic acid, gallic acid, lycopene, glycolic acid, pyrogallol, catechol and muconic acid, which can be used as flavors, cosmetics, sanitizers and animal feed and in pharmacy, among other uses [78,79]. 3.1.1. Enzymes Involved in the PET Degradation Pathway The reason why PET can be degraded by enzymes is because a lot of them are unspecific regarding their substrates. The current literature offers many names given to these enzymes, such as PET hydrolases, PET esterases, PET cutinases, PET depolymerases or PETases. PET esterases, PET cutinases and PET depolymerases are hydrolytic enzymes that Int. J. Mol. Sci. 2024,25, 5536 8 of 22 are able to break the ester bonds of biological molecules, like suberin and cutin, and due to their unspecificity, it turns out that they can break those of PET as well. On the other hand, PETases are hydrolytic enzymes more specific to PET [ 80 , 81 ]. Due to the specificity, there are small differences between the different types of enzymes, such as PET cutinases, which can hydrolyze the ester bonds of aliphatic and aromatic molecules, while PETases can only hydrolyze bonds of aromatic molecules [ 54 , 65 , 69 ]. To simplify the following explanation, we will refer to all of them as PETases unless it is otherwise stated, with the understanding that PETases are enzymes that hydrolyze PET. The hydrolyzation pathway of PET is summarized in Figure 4. In most cases, PETases are capable by themselves of firstly depolymerizing the PET chain and secondly degrading its monomers—mono(2-hydroxyethyl) terephthalate (MHET) and BHET—to finally form TPA and ethylene glycol. PETases can directly produce MHET and TPA, or In contrast, if BHET is generated, they can degrade it to MHET [ 5 , 78 ]. However, there are some PETases that are not able to carry out both steps by themselves, as is the case with PE-H (Pseudomonas aestusnigri) and IsPETase (Ideonella sakaiensis). Both can only depolymerize PET because they cannot break the ester bond of MHET, which accumulates [ 82 ]. In contrast, there are enzymes capable of degrading only MHET (MHETases). IsMHETase is an example, being able to degrade MHET to finally produce ethylene glycol and TPA. Although there are enzymes that do not carry out both degradation actions by themselves, they can complement each other. IsMHETase and IsPETase were found in I. sakaiensis 201-F6 when this strain was discovered in a medium whose only carbon source was PET [ 81 ]. Recently, a new category of enzymes has been discovered, BHETases, which specifically catalyze the transition from BHET to MHET. Two BHETases have been characterized, ChryBHETase from Chryseobacterium sp. and BsEst from Bacillus subtilis [83]. As a consequence of the cooperation between PETases and MHETases or the action of only PETases, PET is hydrolyzed, and TPA and ethylene glycol are released, and they can be metabolized (Figure 4). TPA presents a biochemical pathway to finally be degraded to succinyl-CoA. Regarding ethylene glycol, it can be metabolized to obtain acetyl-CoA [ 78 ]. Both succinyl-CoA and acetyl-CoA are incorporated into the tricarboxylic acid cycle (TCA) to obtain energy for the bacteria and, in certain cases, some products of interest. For example, species I. sakaiensis and Geobacter sulfurreducens when cocultivated can degrade PET to ethylene glycol and generate electricity [ 84 ]. In another study, a Rhodococcus josii strain PET metabolized the PET hydrolysate and synthesized lycopene, which is used in medicine for cancer treatments [ 85 ]. One example of a modified bacteria is Escherichia coli modified to degrade PET and produce vanillin, present in cosmetic and food industries [ 70 ]. The cleavage of the ester bond happens inside the enzyme’s active site, and the ability of different enzymes to hydrolyze PET comes as a consequence of a nucleophilic similarity produced by three amino acids conserved in PETases, Ser-His-Asp. In contrast, the adjacent sequences to these amino acids are different among enzymes, varying their selectivity to substrates [50,63]. There are two proposed classifications for PETases in the literature. One of them is based on their sequence, while the other one is based on their protein structure. According to the sequence-based classification, PETases can be classified as type I and type II enzymes [ 5 ]. Type I enzymes have one C-terminal disulfide bridge, and type II enzymes have two, with one of them being close to the active site. This additional disulfide bridge provides type II enzymes more thermal stability and plays a key role in the hydrolytic action [ 5 , 63 , 86 ]. Another characteristic that differentiates both types is the amino acids present in the active site. Type I enzymes, such as LCC and Cut190, which will be mentioned later, have His159 and Phe/Tyr238 residues, and type II enzymes present Trp and Ser in the same locations. In addition, type II enzymes are subdivided into type IIa if they have Phe or Tyr residues instead of Ser238 or type IIb if they maintain Ser, as is the case with IsPETase. As an exception, type I enzymes may not have the disulfide bridge. This is the case with PET27 and PET30 from Aequorivita sp. CIP111184 and Kaistella jeonii, respectively. According to the structure-based classification, PETases can have three different structures, Int. J. Mol. Sci. 2024,25, 5536 9 of 22 with IsPETase, Fusarium oxysporum cutinase and Chloroflexus sp. MS-G cutinase representing Structures 1, 2 and 3, respectively [ 87 ]. For example, IsPETase presents an α / β -hydrolase fold and a core made up of eight β -strands and six α -helices, as well as a highly polarized surface [65]. Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 of 23 specificity, there are small differences between the different types of enzymes, such as PET cutinases, which can hydrolyze the ester bonds of aliphatic and aromatic molecules, while PETases can only hydrolyze bonds of aromatic molecules [54,65,69]. To simplify the following explanation, we will refer to all of them as PETases unless it is otherwise stated, with the understanding that PETases are enzymes that hydrolyze PET. The hydrolyzation pathway of PET is summarized in Figure 4. In most cases, PETases are capable by themselves of firstly depolymerizing the PET chain and secondly degrading its monomers—mono(2-hydroxyethyl) terephthalate (MHET) and BHET—to finally form TPA and ethylene glycol. PETases can directly produce MHET and TPA, or In contrast, if BHET is generated, they can degrade it to MHET [5,78]. However, there are some PETases that are not able to carry out both steps by themselves, as is the case with PE-H (Pseudomonas aestusnigri) and IsPETase (Ideonella sakaiensis). Both can only depolymerize PET because they cannot break the ester bond of MHET, which accumulates [82]. In contrast, there are enzymes capable of degrading only MHET (MHETases). IsMHETase is an example, being able to degrade MHET to finally produce ethylene glycol and TPA. Although there are enzymes that do not carry out both degradation actions by themselves, they can complement each other. IsMHETase and IsPETase were found in I. sakaiensis 201-F6 when this strain was discovered in a medium whose only carbon source was PET [81]. Recently, a new category of enzymes has been discovered, BHETases, which specifically catalyze the transition from BHET to MHET. Two BHETases have been characterized, ChryBHETase from Chryseobacterium sp. and BsEst from Bacillus subtilis [83]. Figure 4. Chemical structures of PET and products related to its hydrolyzation. Each arrow represents an enzymatic reaction. The presence of multiple arrows, like between EG—acetyl-CoA Figure 4. Chemical structures of PET and products related to its hydrolyzation. Each arrow represents an enzymatic reaction. The presence of multiple arrows, like between EG—acetyl-CoA and TPA— succinyl-CoA, refers to the fact that there is more than one chemical reaction between one molecule and another. Adapted from [80]. 3.1.2. Modifications of Bacteria and Enzymes to Improve PET Degradation Since the discovery of PET-degrading enzymes in the last century, efforts have been made to improve their degradation rate, either by searching for new enzymes and heterologously expressing them in other microorganisms or by modifying the existing ones. It was not until 2016 that the first PETase (IsPETase) was discovered in I. sakaiensis 201-F6, a specific enzyme that allowed the bacteria to use PET as its major carbon and energy source. The experiments were carried out at 30 ◦ C and pH 7, and this enzyme was compared with three other enzymes with hydrolytic activity toward PET [ 81 ]. IsPETase has an α / β hydrolase fold like cutinases but with a larger active site [ 65 ]. After this discovery, modifications of this IsPETase have been carried out in order to obtain a higher degradation rate. The importance of the amino acids of the active site or close to it is emphasized, as will be seen afterward with cutinases. In addition, the presence of one amino acid or another may affect the enzyme–substrate interaction affinity and, therefore, the degradation of the polymer [86]. Int. J. Mol. Sci. 2024,25, 5536 16 of 22 but using a wider variety of polymers could indicate if the modifications have a greater effect than expected and may be more useful. In addition, there is little work on the tridimensional structure of the different enzymes— native or modified—that have been mentioned. Deeper research is necessary on this matter in order to better understand why changes happen regarding the affinity for the substrate or the stability, among other characteristics, according to the exchanged residues. Obtaining the structure of enzymes can be achieved not only through X-ray crystallography but also in silico by modeling. This is especially useful when proteins cannot be successfully purified and crystallized, but modeling goes even further. From molecular dynamics to substrate docking, these tools are used in some of the referenced studies to analyze enzymes’ properties and enhance their activities. Similar to plastics themselves, approaches to studying their biodegradation are diverse, and this variety often comes with a lack of consistency between studies. The conditions in which the enzymes are used can differ, and some studies do not calculate enzymatic activity and limit themselves to detecting solid plastic disintegration or monomer liberation, and the works that calculate enzymatic activity can use different units. This can sometimes make it complicated to compare their conclusions. Overall, more research is required to develop effective, i.e., quicker, safer and more efficient, biodegradation strategies for plastics, if possible. This applies not just to the plastics that are briefly addressed in this work but also to all plastics in general. Microbial biotechnology and genetic engineering approaches, together with the current development of artificial intelligence tools that provide a new direction in the study and design of novel of enzymes, can facilitate the generation and optimization of several types of plastic-degrading enzymes and valorization processes. Nowadays, we are getting closer to achieving this aim thanks to the latest advances in DNA sequencing, metagenomics, bioinformatics, genome mining and machine learning tools, in conjunction with new genetic engineering techniques, such as CRISPR-Cas technologies. Author Contributions: All authors participated in the writing of the work. F.S.-B. edited and handled the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: We acknowledge Laura Andrea Silva López for helping with the representation of the molecular structures shown in Figures 3A and 4. 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